Cells in an organism, regardless of their function, contain identical genetic material yet vary greatly in gene expression and phenotype. Gene transcription is controlled in part through the architecture of its chromatin and through recruitment of transcription factors to specific regulatory elements. These mechanisms are regulated through covalent modifications of DNA and histone proteins that leave the underlying DNA sequence unaltered. Posttranslational modifications of histone proteins are mediated by enzymes that can add or subtract covalent attachments at specific residues. Histones can be methylated, acetylated, phosphorylated, or ubiquitinated and, depending on the residue being modified, identical chemical modifications can have opposing consequences. Histone methyl transferases (HMTs) are enzymes that catalyze the transfer of methyl groups from S-Adenosyl-L-Methionine (SAM) to specific lysine residues of proteins.
Enhancer of Zeste 2 (EZH2) is a HMT that catalyzes methylation of H3K27. Along with cofactors SUZ12, EED, and RbAp46/48, EZH2 forms the Polycomb Repressive Complex 2 (PRC2) (Morey L et al, Trends Biochem Sci 2010; 35 p 323-32.). EZH2 is overexpressed in a wide range of cancers, including advanced-stage and high-grade prostate, breast, and lung tumors (Albert M. et. al., Semin Cell Dev Biol, 2010, 21, p 209-20). EZH2 and PRC2 are critical for the control of gene expression in embryonic stem cells, maintaining self-renewal while inhibiting differentiation (Bracken A P, Genes Dev 2006; 20, p 1123-36), and these properties of EZH2 appear active when the gene is overexpressed in tumors. EZH2 overexpression induces cell migration and colony formation and induces genomic instability by repression of regulators of DNA repair (Kleer C G, et al. Proc Natl Acad Sci USA 2003, 100, p 11606-11.). Conversely, EZH2 depletion suppresses proliferation and attenuates tumor formation in vivo (Gonzalez M E, et al., Oncogene, 2009, 28, p 843-53.). Recently, somatic mutations and deletions of EZH2 were identified in hematologic malignancies, leading to the gain or loss of EZH2 function. Approximately 30% of diffuse large B-cell lymphomas (DLBCL) and 10% of follicular lymphomas contain a mutation at tyrosine 641 (Y641) within the SET domain, predicted to alter the substrate recognition pocket within the enzyme (Morin R D et al., Nat Genet, 2010, 42, p 181-5.). These mutations are always heterozygous, suggesting that they are either dominant to or cooperate with the wild-type (WT) EZH2 protein. Enzymatic studies showed that WT EZH2 converted unmethylated H3K27 to H3K27me1 and to a lesser extent the me2 and me3 states (Sneeringer C J et al, Proc Natl Acad Sci USA., 2010, 107, p 20980-5). By contrast, EZH2Y641X failed to recognize unmethylated H3K27 but readily converted H3K27me1 (created by WT EZH2) to H3K27me2 or me3. Accordingly, DLBCL cells harboring EZH2Y641X display increased levels of H3K27me3. EZH2 is silenced in resting, mature B cells and is transiently upregulated in germinal center B cells, where, along with BCL6, it blocks DNA damage response pathways allowing cells to survive the somatic hypermutation of antibody maturation (Velichutina I et al Blood 2010, 116, p 5247-55). By amplifying these functions and targeting additional pathways, EZH2 mutations may stimulate malignant transformation. In myeloid neoplasia, EZH2 is most often affected by deletions and nonsense mutations that yield loss of function, and leukemia cell lines harboring EZH2 mutations show decreased H3K27 methylation (Ernst T et al Nat Genet 2010, 42, p 722-6.). The presence of activating and inactivating EZH2 mutations in different cancers suggest a complex, context-dependent role of Polycomb proteins in oncogenesis. It is unclear whether EZH2 affects different sets of genes in different malignancies or whether global histone changes may interfere with other chromatin functions such as replication and DNA repair. Nevertheless, the frequent occurrence of genetic lesions affecting H3K27 suggests that this mark is under tight control, which may present a challenge in the design of safe and effective EZH2 inhibitors. The S-adenosylhomocysteine (SAH) hydrolase inhibitor 3-Deazaneplanocin A (DZNeP) is an early example of an EZH2 inhibitor (Tan J, et al. Genes Dev 2007, 21, p 1050-63.). DZNeP can inhibit HMTs by increasing SAH levels, inducing the degradation of EZH2 and leading to a global decrease of H3K27 methylation accompanied by apoptosis of cancer cells. However, in some cells, DZNeP decreases methylation of multiple other histone residues, perhaps as a result of the ability of SAH to compete with the AdoMet cofactor (Miranda T B et al. Mol Cancer Ther 2009, 8, p 1579-88.). Hence, more specific inhibitors of EZH2 are required to address various lesions in cancer. Recently inhibitors of EZH2, acting as a SAM competitor and affecting specifically the HMT activity of EZH2 (McCabe M T et al, Nature, 2012, 492, p 108-112.) have been reported. They also show antiproliferative activity in DLBCL cell lines expressing mutant EZH2, suggesting that direct inhibition of EZH2 activity presents a promising avenue for treatment in the clinic.